Acute myeloid leukaemia (AML) is a term used to define a heterogeneous group of haematological disorders resulting from the malignant transformation of myeloid precursor cells. Transformation leads to the proliferation of immature and undifferentiated cells in the blood and bone marrow and suppression of normal haemopoiesis. Effective treatment of AML patients is challenging and the clinical outcome can be disappointing and unpredictable. Only 70% of newly diagnosed patients receiving standard regimens respond to treatment. Furthermore, a large proportion of these patients fail to achieve long-term remission and develop resistance to subsequent therapy.
Nucleoside analogues (NA) are a class of antimetabolite drug widely used in the treatment of certain types of cancer and viral infections. These compounds resemble structurally the body's natural nucleosides and are subject to the same physiological uptake and metabolic mechanisms which results in their incorporation into newly synthesised DNA. This DNA-adulteration results in DNA synthesis inhibition and chain termination leading to cell death. NA are prodrugs that require phosphorylation into triphosphates to form the active nucleotide. Certain NA do not disrupt directly DNA replication. For example, the anti-herpes drug acyclovir, following cellular uptake and phosphorylation, disrupts DNA replication by inhibiting DNA polymerase.
The nucleoside analogue Ara-C is one of the most active single anti-cancer agents and has been the mainstay treatment of AML for over three decades. In vivo, Ara-C is transported into the cell via the specific nucleoside transporter hENT1 and is rapidly phosphorylated by dCK to its monophosphate form. Ara-CMP is further phosphorylated by nucleoside kinases into its active tri-phosphorylated form, Ara-CTP. The anti-proliferative and cytotoxic effects of Ara-CTP are due to its ability to interfere with DNA polymerase and to incorporate into DNA strands leading to chain termination and arrest of DNA synthesis. High-dose Ara-C can also cause accumulation of cytochrome C in the cytosol, loss of mitochondrial membrane potential and increase in the reactive oxygen species.
Chemoresistance to Ara-C can arise from a number of factors influencing the rate of Ara-CTP formation and incorporation into DNA, including low drug uptake, conversion into Ara-U by cytidine deaminase, or dephosphorylation of the active metabolite by cytoplasmic nucleotidases.
The primary mechanism of resistance to nucleoside analogue drugs is due to insufficient NA triphosphate. The main causes of this are decreased levels of phosphorylating enzymes (e.g. dCK), inefficient cellular uptake (e.g. decreased levels of hENT1), increased levels of NA degrading enzymes (notably cytidine deaminase, cdd), and expansion of deoxyribonucleotide triphosphate (dNTP) pools. Deoxyribonucleoside kinase (dNK) levels are also considered to play key a role in drug resistance.
Ara-C has no effect on Escherichia coli as it lacks dCK and deaminates Ara-C into Ara-uracil through the activity of deoxycytidine deaminase (cdd). Wang et al., Antimicrob. Agents. Chemother., 1998, 42:2620-2625, described the construction of a cdd-deficient E. coli mutant (SØ5218) which, upon the expression of human dCK gene, exhibited reduced relative growth in the presence of Ara-C. The Ara-C effect on growth was completely abolished when assayed in the absence of the dCK inducible promoter, IPTG, indicating human dCK expression in the bacteria leads to Ara-CTP incorporation into bacterial DNA.
In vitro assessment of Ara-C efficacy has traditionally involved measurement of a) cell death b) reduction in S phase activity or c) use of AML clonogenic assays following exposure of leukaemic cells to Ara-C. These methods are non-standardised, time-consuming, expensive and are not suitable for routine screening. Therefore, patients are treated with regimens including Ara-C regardless of their sensitivity to the drug and can suffer debilitating side-effects such as myelosuppression, nausea, diarrhoea, vomiting and the development of drug-resistant secondary cancers.
Thus there is a need for a simple, rapid pre-screening test for determining nucleoside analogue drug efficacy in patients.